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Preparation And Gene Transfection Properties Of Tumor Targeting Gene Vectors Based On Polyethylenimine Derivative Conjugated With Biotin-Avidin System

Posted on:2011-06-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:X CengFull Text:PDF
GTID:1224360305483250Subject:Polymer Chemistry and Physics
Abstract/Summary:PDF Full Text Request
Gene therapy is a new form of molecular medicine to cure the inherited or acquired diseases through delivering therapeutic genes to targeted cells and replacing the disorder genes. Many diseases could be treated, such as heritable disorders and cancers. Nowadays, substantial progress has been made in cancer gene therapy, and it has been used widely in human clinical trials. As delivery of "naked" DNA to cell is too inefficient, most gene transfer is carried out using a powerful gene delivery vehicle. Among various gene vectors, the non-viral polycations play an important role in gene delivery due to their high safety, simplicity for preparation and adjustable structures. However, current non-viral gene delivery systems still have defects, including lacking tumor-targeted ability, low transfection efficiency, high cytotoxicity and poor serum stability, which directly limited their clinical applications. Hence, the aim of this study is to prepare a series of tumor-targeted non-viral vectors for cancer gene therapy. They will be multifunctional systems, which can give DNA better protection against enzymes, lead to specific gene delivery in tumor cells, enhance endosomal escape, and be more biocompatible or biodegradable.In chapter 1, the definition and barriers of gene therapy were introduced. The current significant non-viral gene delivery systems and therapeutic genes were reviewed.In chapter 2, a novel gene vector biotinylated polyethyleneimine/avidin bioconjugates (ABP) was synthesized by coupling biotin onto the backbone of high molecular weight branched PEI (25 kDa) and bio-conjugating with avidin by the avidin-biotin strong affinity. The resulted gene vector ABP was evaluated in terms of gel retardation assay, particle size,ζ-potential, in vitro cell viability and transfection efficiency. Results indicated that ABP demonstrates significant lower cytotoxicity and higher transfection efficacy in HepG2 cells due to the biocompatibility of avidin and the unknown interaction between avidin and HepG2 cells. ABP obtained here will have the potential in targeting gene delivery of liver cell and could also be useful for other forms of hepatic disease.In chapter 3, biotinylated disulfide containing polyethyleneimine/avidin bioconjugate (ABP-SS) was synthesized as a targeting gene vector, which presented less toxic and higher gene expression, especially in HepG2 cells. This was accomplished by grafting biotin to disulfide-containing polyethylenimine (PEI-SS) and bioconjugating with avidin by the avidin-biotin strong affinity. The resulted gene vector ABP-SS and its pDNA complexes were evaluated in terms of gel retardation assay, SEM, particle size,ζ-potential, in vitro cell viability and transfection efficiency. Results indicated that ABP-SS, compared with PEI-SS, demonstrated significant lower cytotoxicity and much higher transfection efficacy in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells. ABP-SS provided a promising potential in targeting gene delivery of liver cell and could also be useful for other forms of hepatic disease.In chapter 4, in order to confirm the optimum condition for in vitro gene transfection of biotinylated PEI-SS/avidin bioconjugate (ABP-SS)/DNA complexes in HepG2 cells. ABP-SS mediated gene transfection was exposed to various environmental factors, such as pH, NaCl, serum, temperature and time. Physiochemical characteristics of ABP-SS/DNA complexes were evaluated in terms of agarose gel electrophoresis, particle size measurements, and in vitro transfection assay. The DNA binding ability of ABP-SS was weakened when pH value was decreasing. It was inferred that ABP-SS/DNA complexes could form compact structures at pH 7.4 and looser structures at lower pH values. Transfection efficiencies were largely dependent on pH of culture medium, and the optimal pH of culture medium for ABP-SS mediated gene delivery in HepG2 was 7.4. The particle sizes of ABP-SS/pDNA complexes formed in 150 mM NaCl solution were less than 280 nm, and larger than that of complexes formed in deionized water. The transfection ability of ABP-SS/pGL-3 complexes formed in deionized water was much weaker than that formed in 150 mM NaCl solution. The presence of lower serum contents had no obvious effect on the transfection efficiency of ABP-SS/pGL-3 complexes. The optimum incubating temperature and time for ABP-SS/pGL-3 complexes were 37℃and 30min. Cell morphology was observed by live cell confocal microscopy. It can be concluded that cytotoxicity of ABP-SS in HepG2 cells was related to apoptosis.In chapter 5, biotinylated transferrin/avidin/biotinylated disulfide containing PEI bioconjugates (TABP-SS) mediated p53 gene delivery system was formed attributed to the’avidin-biotin bridge’. Characteristics of the obtained TABP-SS and its p53 complexes were evaluated in terms of acid-base titration, agarose gel electrophoresis, SEM, particle size andζ-potential measurements. The acid-base titration results showed that TABP-SS had good buffer capability. The results of gel electrophoresis indicated that TABP-SS could fully condensed DNA and would be degraded by reducing agent inside cells. In vitro cell viability and transfection of TABP-SS were investigated in COS7, HepG2, and HeLa cells. Among the three different cell lines, TABP-SS exhibited much lower cytotoxicity and higher transfection efficacy in HepG2 and HeLa cells due to the specific interactions between transferrin ligands and their receptors on tumor cells. Apoptotic morphology was observed using confocal microscopy, and the expression of p53 protein in transfected cells was evaluated by western blotting. All the results indicated that TABP-SS/p53 complex could be considered as a low toxic and high efficient tumor targeted gene delivery system, which has great potential for further clinical application.
Keywords/Search Tags:gene therapy, non-viral vectors, polyethylenimine, biotin-avidin system, tumor targeting
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